Cytosolic phospholipase A2 (cPLA2) is a key regulatory enzyme that mediates agonist-induced arachidonic acid release. Arachidonic acid itself is an important intracellular regulator and can also be converted to a variety of potent lipid metabolites, the eicosanoids. This occurs through the lipoxygenase and cyclooxygenase pathways for the production of leukotrienes, and prostaglandins. These oxygenated metabolites of arachidonic acid play crucial roles in normal homeostasis and in mediating the inflammatory response. Intracellular levels of arachidonic acid are tightly controlled and cPLA2 is subject to complex mechanisms of activation that are poorly understood. cPLA2 is post-translationally regulated by phosphorylation and by levels of intracellular calcium. Agonist-induced serine phosphorylation of cPLA2 on Ser505 by mitogen activated protein kinase enhances cPLA2 activity. An increase in intracellular calcium triggers cPLA2 activation by inducing its translocation from cytosol to the nuclear envelope/endoplasmic reticulum (ER), where 5-lipoxygenase and cyclooxygenase are also localized. Calcium-induced binding of cPLA2 to membrane is mediated by the calcium-dependent phospholipid binding domain (CaLB or C2 domain) on the amino terminus of cPLA2. Although the role of the CaLB domain in membrane binding is established, the mechanisms involved in the specific targeting of cPLA2 to nuclear membrane/ER are not known, and their elucidation is the main objective of this proposal. The specific aims of the proposal include defining the role of the C2 domain and regions downstream of the C2 domain (including the plekstrin homology domain of cPLA2) in regulating nuclear targeting, and identifying proteins that bind cPLA2 and determining their role in regulating cPLA2 activity and translocation. Potential cPLA2 binding proteins have been identified using a biochemical approach and screening a phage display library. Domains of cPLA2 that are important in regulating nuclear targeting will be identified using site-directed mutagenesis. Effects of the mutations on agonist-induced arachidonic acid release and translocation of cPLA2 to the nucleus will be evaluated by expression in mammalian cells and in insect cells using baculovirus. Purified mutant constructs will also be evaluated for effects on enzymatic activity and lipid vesicle binding. Understanding the regulation of cPLA2 will provide greater insight into the signal transduction mechanisms involved in lipid mediator production, and lead to potential methods for pharmacological modulation of the inflammatory process.